To truly test our understanding of complex biomolecular systems and have complete control over the behavior of constituent parts, we must learn how to build integrated biological processes from scratch. The long-term vision of this project is to construct a synthetic replicating entity from small molecule building blocks. Self-replication from small molecules should be enabled by reconstitution of three biochemical systems from "the central dogma": DNA, RNA, and protein syntheses. Cell-free technologies have been critical in elucidating fundamental principles that impact our ability to diagnose, treat, and prevent disease (such as Nirenberg discovering the genetic code). The benefits of creating this cell-free molecular chassis are enormous. First, construction efforts will enhance our understanding of biological self-assembly. This will lead to new antibiotic targets for addressing growing health concerns over rising bacterial resistance. Second, a standardized cell-free scaffold will offer an exquisite platform for studying the central machinery of life. This will help address the tremendous need for testing hypotheses that have arisen from advances in genome sequencing technology. Third, made-to-order cell-free synthetic factories will serve as a basic starting point for robust engineering efforts to produce protein therapeutics and peptide drugs. A minimal genome for the synthetic replicon has been defined from Escherichia coli comprising 151 genes. Toward the vision of constructing this biological machine, the specific aims are to: (1) Synthesize proteins encoded by the minimal genome using cell-free translation. We will demonstrate that the constituent parts (both individually and as operons) can be made in cell-free systems. (2) Develop a cell-free method for synthesis and assembly of an active E. coli ribosome. Here we will learn how to build a ribosome from parts made entirely in vitro using a strategy that moves from semi-synthetic to entirely synthetic parts. Translation efficiency and fidelity of reconstituted subunits will be tested. Insights to new drug targets are expected. (3) Engineer metabolic modules for supplying energy to the synthetic replicating entity. Because fueling cell-free reactions is a known limitation and we would also like to be able to add functionality to the main chassis, oxidative phosphorylation and glycolysis modules will be created and characterized.